Identification of novel mutations in hemochromatosis genes by targeted next generation sequencing in Italian patients with unexplained iron overload

Abstract

Hereditary hemochromatosis, one of the commonest genetic disorder in Caucasians, is mainly associated to homozygosity for the C282Y mutation in the HFE gene, which is highly prevalent (allele frequency up to near 10% in Northern Europe) and easily detectable through a widely available "first level" molecular test. However, in certain geographical regions like the Mediterranean area, up to 30% of patients with a HH phenotype has a negative or non-diagnostic (i.e. simple heterozygosity) test, because of a known heterogeneity involving at least four other genes (HAMP, HJV, TFR2, and SLC40A1). Mutations in such genes are generally rare/private, making the diagnosis of atypical HH essentially a matter of exclusion in clinical practice (from here the term of "non-HFE" HH), unless cumbersome traditional sequencing is applied. We developed a Next Generation Sequencing (NGS)-based test targeting the five HH genes, and applied it to patients with clinically relevant iron overload (IO) and a non-diagnostic first level genetic test. We identified several mutations, some of which were novel (i.e. HFE W163X, HAMP R59X, and TFR2 D555N) and allowed molecular reclassification of "non-HFE" HH clinical diagnosis, particularly in some highly selected IO patients without concurring acquired risk factors. This NGS-based "second level" genetic test may represent a useful tool for molecular diagnosis of HH in patients in whom HH phenotype remains unexplained after the search of common HFE mutations.

Identification of new BMP6 pro‐peptide mutations in patients with iron overload

“…Our recently described Next‐Generation Sequencing (NGS)‐based test for IO disorders, updated to include the BMP6 gene (see below for details), was applied to 37 probands (mean age 51.5 ±15.0 years, 89% males), with a putative diagnosis of atypical IO due to hyperferritinemia and increased Liver Iron Content (LIC > 36 µmol/g) detected by magnetic resonance imaging (MRI), as well as a negative or nondiagnostic first level genetic test for HH (i.e., neither p.Cys282Tyr homozygosity nor p.Cys282Tyr/p.His63Asp compound heterozygosity in the HFE gene). Four patients out of this series were found to carry a BMP6 heterozygous mutation.…”

“…All of the established HH genes are involved in the production and function of the liver hormone hepcidin, the key regulator of systemic iron homeostasis . Notwithstanding the great advances in the molecular diagnosis of HH recently achieved by targeted sequencing, some patients with HH‐like phenotypes do not carry mutations in the known genes, suggesting the involvement of still unknown genetic factors. Among others, Bone Morphogenetic Proteins (BMPs) represent compelling candidates.…”

Clinical evaluation of a hemochromatosis next‐generation sequencing gene panel

“…Whole-exome sequencing has been employed in small cohorts of patients with extreme iron overload identified modulating mutations (13), targeted sequencing of eight patients with iron overload (12), and in a family with evidence of extreme iron overload resulting from macrocytic dyserythropoietic anemia (14). Targeted gene panels and next-generation sequencing identified non-HFE mutations in small cohorts have also been reported (15,16). Our hypothesis was that sequencing 15 iron metabolism genes using a targeted next-generation sequencing platform would assist in the molecular diagnosis of patients with suspected iron overload in our clinically ascertained cohort.…”

Next-generation sequencing of hereditary hemochromatosis-related genes: Novel likely pathogenic variants found in the Portuguese population

“…As far as we know, only two studies have been published concerning the application of NGS to atypical iron disorders [33,34]. The identification and study of novel iron metabolism-related mutations are important steps forward to improve the knowledge of the HH genetic basis heterogeneity and of the pathophysiology of the different types of HH.…”